This section is intended to introduce the reader to various aspects of art that may be related to various aspects of the present invention, which are described and/or claimed below. This discussion is believed to be helpful in providing the reader with background information to facilitate a better understanding of various aspects of the present invention. Accordingly, it should be understood that these statements are to be read in this light, and not as admissions of prior art.
Since its initial design by Kary Mullis in 1984, the polymerase chain reaction (PCR) has impacted nearly every field in molecular biology, genetics, and forensic science. PCR is a technique to amplify a single or few copies of a particular nucleic acid sequence, e.g., DNA (the target DNA sequence), across several orders of magnitude, thereby generating thousands to millions of copies of the sequence. PCR is the main tool presently used to amplify nucleic acid and study gene expression.
PCR relies on “thermal cycling,” which includes cycles of repeated heating and cooling of the DNA and other reaction components to cause DNA denaturation (i.e., separation of the double-stranded DNA into its sense and antisense strands) followed by enzymatic replication of the DNA. The other reaction components include short oligonucleotide DNA fragments known as “primers,” which contain sequences complementary to at least a portion of the target DNA sequence, and a DNA polymerase. These are components that facilitate selective and repeated amplification of the target sequence. As PCR progresses, the DNA generated is itself used as a template for further replication in subsequent cycles, creating a chain reaction in which the target DNA sequence is exponentially amplified.
More specifically, the DNA polymerase used in PCR is thermostable (and thus avoids enzyme denaturation at high temperatures) and amplifies target DNA by in vitro enzymatic replication. One such thermostable DNA polymerase is Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. The DNA polymerase enzymatically assembles a new DNA strand from deoxynucleoside triphosphates (dNTPs) by using the denatured single-stranded DNA as a template. As is known to those of ordinary skill in the art, a deoxynucleoside triphosphate is deoxyribose) having three phosphate groups attached, and having one base (adenine, guanine, cytosine, thymine) attached. However, as used herein, it will be recognized by those of ordinary skill in the art that arsenic may be substituted for phosphorous in the triphosphate back bone any dNTP. The initiation of DNA synthesis and the selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.
Thus, a basic PCR set up includes multiple components. These include: (1) a DNA template that contains the target DNA region to be amplified; (2) primers that are complementary to the 3′ ends of each of the sense strand and anti-sense strand of the target DNA; (3) a thermostable DNA polymerase such as Taq polymerase; and (4) dNTPs, the building blocks from which the DNA polymerases synthesizes a new DNA strand. Additionally, the reaction will generally include other components such as a buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerase, divalent cations (generally magnesium ions), and monovalent cation potassium ions (K+).
PCR is commonly carried out in a reaction volume of 10-200 μl in small reaction tubes (0.2-0.5 ml volumes) in an apparatus referred to as a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each of the following steps of the reaction:
Denaturation Step:
This step consists of heating the reaction to usually around 94-98° C. for approximately 20-30 seconds. It causes denaturation of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single strands of DNA.
Annealing Step:
The reaction temperature is lowered to usually around 50-65° C. for approximately 20-40 seconds allowing annealing of the primers to the single-stranded DNA template. Stable DNA-DNA hydrogen bonds are formed when the primer sequence closely matches the template sequence. The polymerase (e.g., Taq polymerase) binds to the primer-template hybrid and begins DNA synthesis.
Extension Step:
The temperature at this step depends on the DNA polymerase used. Taq polymerase has its optimum activity temperature at about 75° C., and commonly a temperature of 72° C. is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in the 5′ to 3′ direction, condensing the 5′-phosphate group of the dNTPs with the 3′-hydroxyl group at the end of the extending DNA strand. The extension time depends on the DNA polymerase used and on the length of the DNA fragment to be amplified. Under optimum conditions, at each extension step the amount of the target DNA is doubled, leading to exponential amplification of the specific target DNA.
PCR usually includes of a series of 20 to 40 repeated cycles of the above-described denaturation, annealing, and extension steps. The cycling is often preceded by a single initialization step at a high temperature (>90° C.), and followed by one final hold at the end for final product extension or brief storage. The initialization step consists of heating the reaction to a temperature of usually 94-96° C. (or 98° C. if extremely thermostable polymerases are used), which is held for 1-9 minutes. The final hold usually occurs at 4-15° C. for an indefinite time and may be employed for short-term storage of the reaction. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.
Following thermal cycling, agarose gel electrophoresis may be employed for size separation of the PCR products to check whether PCR amplified the target DNA fragment. The size(s) of the PCR products is determined by comparison with a molecular weight marker, which contains DNA fragments of known size, run on the gel alongside the PCR products.
There are many applications of PCR. For example, real-time PCR (RT-PCR) is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification. Thus, RT-PCR enables both detection and quantification of one or more specific sequences in a DNA sample (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes). Such quantitative PCR methods allow the estimation of the amount of a given sequence present in a sample—a technique often applied to quantitatively determine levels of gene expression.
RT-PCR procedure follows the general principle of PCR. However, in RT-PCR, the amplified DNA is detected as the reaction progresses in real time (whereas in standard PCR, the product of the reaction is detected at the end of the reaction). One common method for detection of products in RT-PCR is the use of nonspecific fluorescent dyes that intercalate with double-stranded DNA (dsDNA). For example, SYBR Green is an asymmetrical cyanine dye that binds to dsDNA, and the resulting DNA-dye complex absorbs blue light (λmax=488 nm) and emits green light (λmax=522 nm). The DNA-binding dye, such as SYBR Green, binds to all dsDNA in PCR, causing fluorescence of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity and is measured at each cycle, thus allowing DNA concentrations to be quantified.
Another method for detection of products in RT-PCR is the use of sequence-specific DNA probes, which are oligonucleotides that are labeled with a fluorescent reporter that permits detection after hybridization of the probe with its complementary DNA target. Many of these probes include a DNA-based probe having a fluorescent reporter (e.g., at one end of the probe) and a quencher of fluorescence (e.g., at the opposite end of the probe). The close proximity of the reporter to the quencher prevents detection of its fluorescence; breakdown of the probe by the 5′ to 3′ exonuclease activity of the Taq polymerase breaks the reporter-quencher proximity and thus allows unquenched emission of fluorescence, which can be detected. An increase in the product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of the probe and release of the reporter. Examples of such probes well known to those of ordinary skill in the art are molecular beacon probes, TagMan® probes, and Scorpion™ probes.
Molecular beacons are single-stranded oligonucleotide probes that form a hairpin-shaped stem-loop structure. The loop contains a probe sequence that is complementary to a target sequence in the PCR product. The stem is formed by the annealing of complementary sequences that are located on either side of the probe sequence. A fluorophore and quencher are covalently linked to the ends of the hairpin. Upon hybridization to a target sequence the fluorophore is separated from the quencher and fluorescence increases. Hybridization usually occurs after unfolding of the hairpin and product duplexes in the denaturation step of the next PCR cycle.
TaqMan® probes are single-stranded unstructured oligonucleotides. They have a fluorophore attached to the 5′ end and a quencher attached to the 3′ end. When the probes are free in solution, or hybridized to a target, the proximity of the fluorophore and quencher molecules quenches the fluorescence. During PCR, when the polymerase replicates a template on which a TagMan® probe is bound, the 5′-nuclease activity of the polymerase cleaves the probe. Upon cleavage, the fluorophore is released and fluorescence increases.
Scorpion™ probes use a single oligonucleotide that consists of a hybridization probe (stem-loop structure similar to molecular beacons) and a primer linked together via a non-amplifiable monomer. The hairpin loop contains a specific sequence that is complementary to the extension product of the primer. After extension of the primer during the extension step of a PCR cycle, the specific probe sequence is able to hybridize to its complement within the extended portion when the complementary strands are separated during the denaturation step of the subsequent PCR cycle, and fluorescence will thus be increased (in the same manner as molecular beacons).
However, there are drawbacks to the probes used to detect products in RT-PCR. For example, in the case of the use of nonspecific fluorescent dyes, dsDNA dyes such as SYBR Green will bind to all dsDNA PCR products, including nonspecific PCR products (such as “primer dimers”—i.e., primer molecules that have hybridized to each other). This interferes with and prevents accurate quantification of the intended target DNA sequence.
The use of sequence-specific DNA probes (e.g., molecular beacons, TagMan®, and Scorpion™ probes) reduces or eliminates some of the drawbacks inherent in nonspecific fluorescent dyes. For example, fluorescent reporter probes detect only the DNA containing the probe sequence; therefore, use of the reporter probe significantly increases specificity, and enables quantification even in the presence of non-specific DNA amplification. Fluorescent probes can be used in multiplex assays—for detection of several genes in the same reaction—based on specific probes with different-colored labels, provided that all targeted genes are amplified with similar efficiency. The specificity of fluorescent reporter probes also prevents interference of measurements caused by primer dimers.
However, there are also drawbacks with these sequence-specific probes. For example, there are several disadvantages with molecular beacons. First, they require two bulky and costly tags (fluorophore and quencher). Second, the assay requires a separate probe for each template (i.e. mRNA), which dramatically increases the design effort and expense. Third, the mechanism uses separate binding sites for primer and probe sequences, which introduces another component (probe oligonucleotide) to an already complex reaction, and adds additional design limitations due to the need to avoid interactions between the probe and primers. Fourth, hybridization of the probe requires heating steps to unfold the product duplex and hairpin. Consequently, molecular beacons can't be used under isothermal conditions. Fifth, design of the probe requires considerable effort and knowledge of nucleic acid thermodynamics. And sixth, probe hybridization involves a bimolecular probe-primer system. This makes the reaction entropically unfavorable, slows down hybridization, and complicates product detection at exponential growth. The hybridization is much faster and efficient with a monomolecular probe-primer system [as described in Whitcombe, D. et al. (1999) Detection of PCR products using self-probing amplicons and fluorescence. Nat Biotechnol, 17, 804-807, incorporated by reference herein in its entirety].
All of the shortcomings listed above for molecular beacons hold true for TaqMan® probes. And, an additional disadvantage of TaqMan® probes is that they require the 5′-nuclease activity of the DNA polymerase used for PCR.
Additionally, many of the shortcomings listed for molecular beacons hold true for Scorpion™ probes. First, they require two bulky and costly tags (fluorophore and quencher). Second, the assay requires a separate probe for each template (i.e. mRNA), which dramatically increases the design effort and expense. Third, the mechanism uses separate binding sites for primer and probe sequences, which introduces another component (probe oligonucleotide) to an already complex reaction, and adds additional design limitations due to the need to avoid interactions between the probe and primers. Fourth, hybridization of the probe requires heating steps to unfold the product duplex and hairpin. Consequently, molecular beacons can't be used under isothermal conditions. And fifth, design of the probes requires considerable effort and knowledge of nucleic acid thermodynamics.
Further, fluorescent reporter probes do not prevent the inhibitory effect of the primer dimers, which may depress accumulation of the desired products in the reaction.
Apart from any problems with probes listed above, there are further problems inherent in PCR and RT-PCR. For example, one of the most important factors limiting the yield of specific product is the competition between primer binding and self-annealing of the product. At the initial stage of PCR, product molecules are at low enough concentrations that product self-annealing does not compete with primer binding and amplification proceeds at an exponential rate. However, with accumulation of product DNA, self-annealing becomes dominant and PCR slows down and eventually DNA amplification ceases.
Temperature cycling is another limitation of PCR since it requires expensive instrumentation for thermocycling and complicates rapid detection of pathogens in the field and at point-of-care. In addition, rapid temperature changes facilitate product mis-priming and affect stability of the polymerases.
To decrease certain of the above-described drawbacks, such as the cost of probe synthesis, several attempts have been made to use intrinsic fluorescence of nucleotides. For example, 2-aminopurine (2Ap) has been used as a label for the detection of product in RT-PCR. 2Ap is a fluorescent analog of adenosine and has been used as a site-specific probe of nucleic acid structure and dynamics because it base pairs with cytosine in a wobble configuration or with thymine in a Watson-Crick geometry. 2Ap has been incorporated into stem-loop probes [as described in Walker, G. T. et al. (1992) Strand displacement amplification—an isothermal, in vitro DNA amplification technique. Nucleic acids research, 20, 1691-1696]. Upon hybridization to a target sequence, a several-fold increase in fluorescence was observed due to the change of 2Ap from a double-helix (quenched state) to a single-stranded region (emitted state). However, the sensitivity of the 2Ap probes is insufficient for accurate monitoring of PCR, since 2Ap is still significantly quenched in single strands.